Abstract
Plasmid R6K, which contains 3 replication origins called alpha, gamma, and beta, is a favorable system to investigate the molecular mechanism(s) of action at a distance, i.e. replication initiation at a considerable distance from the primary initiator protein binding sites (iterons). The centrally located gamma origin contains 7 iterons that bind to the plasmid-encoded initiator protein, pi. Ori alpha, located at a distance of approximately 4 kb from gamma, contains a single iteron that does not directly bind to pi but is believed to access the protein by pi-mediated alpha-gamma iteron-iteron interaction that loops out the intervening approximately 3.7 kb of DNA. Although the cis-acting components and the trans-acting proteins required for ori gamma function have been analyzed in detail, such information was lacking for ori alpha. Here, we have identified both the sequence elements located at alpha and those at gamma, that together promoted alpha activity. The data support the conclusion that besides the single iteron, a neighboring DNA primase recognition element called G site is essential for alpha-directed plasmid maintenance. Sequences preceding the iteron and immediately following the G site, although not absolutely necessary, appear to play a role in efficient plasmid maintenance. In addition, while both dnaA1 and dnaA2 boxes that bind to DnaA protein and are located at gamma were essential for alpha activity, only dnaA2 was required for initiation at gamma. Mutations in the AT-rich region of gamma also abolished alpha function. These results are consistent with the interpretation that a protein-DNA complex consisting of pi and DnaA forms at gamma and activates alpha at a distance by DNA looping.